This project addresses an emerging demand for long-term, high content, three-dimensional intra- incubator imaging of multilayer specimens such as three dimensional (3D) organ models of disease progression or engineered replacement tissues developing under tight environmental conditions. With these types of tissue constructs in mind, we propose to design and build a compact and inexpensive- to-replicate multi-color confocal imaging system that operates within a standard CO2 or CO2/O2 controlled incubator to allow for days or weeks of 3D image data to be collected at multiple positions on an XY stage. We will evaluate and verify our instrument's unique functionality by carrying out extended imaging experiments on a microfabricated in vitro cancer model system designed to recapitulate tumor-induced endothelial sprouting. Three-dimensional in vitro cultures have been shown to better recapitulate the physiological microenvironment than 2D models, providing highly controllable experimental parameters in a more in vivo-like system. These types of experimental systems are an important middle ground between simpler 2D monolayers of cells and more complex animal imaging experiments, and are an excellent example of the types of studies that would benefit the most from the novel approach described here. Currently, analysis of the underlying cellular and molecular phenomena is usually limited to low sample numbers and single time point measurements. Using the instrument developed in this project we will show that we can perform long-term, continuous studies of tumor angiogenesis, cellular motility and interactions, and the efficacy of anti-angiogenic therapies. By the end of the funding period we will have created and demonstrated a new imaging platform that will be ready for commercial development so that the unique technology we design is disseminated to the entire biomedical community. This type of instrument would find important uses in many areas of biomedical research in addition to the validation study we propose here. The instrument design described here will become a critical research tool for long term high resolution observations of model systems of disease progression, drug efficacy studies and development of engineered replacement tissues such as cartilage or heart values, which will lead to an improved understanding of disease and help improve human health.